Phylogeny of Canadian ergot fungi and a detection assay by real-time polymerase chain reaction

ABSTRACT

The ergot disease of cereals has become increasingly important in agricultural areas of Canada since 1999. Generally, this disease is considered to be caused by Claviceps purpurea, but the taxonomy of Claviceps from these areas has not been well studied. The objectives of this study were (i) to determine the phylogenetic lineages (phylogenetic species) present in agricultural areas of Canada and (ii) to develop a molecular assay that can separate the lineages on crops from other lineages. Genetic diversity of Claviceps collected from agriculture areas in Canada were investigated using multilocus sequence typing. The loci sequenced include nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), partial fragments of translation elongation factor 1-α (TEF1), RNA polymerase II second largest subunit (RPB2), β-tubulin (tubB), and two ergot alkaloid synthesis genes (easA, easE). Based on individual locus and concatenated alignments, phylogenetic analyses revealed seven lineages within the premolecular concept of C. purpurea, of which five corresponded with undescribed species (G2b and G4–7). Although lineages G2–7 had narrow host ranges, lineage G1 (= C. purpurea s.s.) had a broad host range that overlapped with other lineages. A molecular diagnostic quantitative polymerase chain reaction (qPCR) assay was developed and validated with 185 samples from a wide range of host plants and geographic origins, including 10 phylogenetic species in C. sect. Claviceps, 8 in C. sect. Pusillae, 1 in C. sect. Citrinae, and 1–2 species from Alternaria, Fusarium, and Penicillium. The assay can detect lineage G1 at a concentration of 7.5 pg/μL and distinguish it from other Claviceps species and lineages. This facilitates disease management by detecting the inocula from nonagriculture host plants.

KEYWORDS:

• Ascomycota
• cereal disease
• Claviceps purpurea
• DNA bar-coding
• Hypocreales
• molecular diagnostic
• phylogenetic species

ACKNOWLEDGMENTS

We thank Nicole Beck, Jason Liang, Maria Mankel, Olivia C. Pochopsky, and the Molecular Technologies Laboratory (MTL) at the Ottawa Research and Development Centre, specifically Julie T. Chapados, Kasia Dadej, Wayne McCormick, and Lisa Koziol, for technical assistance. We thank Canadian Collection of Fungal Cultures (CCFC), Canadian National Mycological Herbarium (DAOM), and Drs. Miroslav Kolařík (Culture Collection of Clavicipitaceae, CZ), Stephen Darbyshire, and Jacques Cayouette for providing ergot samples or cultures. Drs. Guillaume Bilodeau, Tom Graefenhan, and Sarah Hambleton, Ms. Tara Rintoul, and two anonymous reviewers provided valuable advice for drafts of the manuscript.

Supplemental data

Supplemental data for this article can be accessed on the publisher’s Web site.

Additional information

Funding

The study was funded by Agriculture and Agri-Food Canada’s Growing Forward 2 for a research network on Emerging Mycotoxins (EmTox, project no. J-000048).